J Biochem Mol Biol, 2002 Jul 31, 35(4), 437 - 41 Activity of human dihydrolipoamide dehydrogenase is reduced by mutation at threonine-44 of FAD-binding region to valine; Kim H; Dihydrolipoamide dehydrogenase (E3) is a member of the pyridine nucleotide-disulfide oxidoreductase family . Thr residues are highly conserved . They are at the active site disulfide-bond regions of most E3s and other oxidoreductases . The crystal structure of Azotobacter vinelandii E3 suggests that the hydroxyl group of Thr that are involved in the FAD binding interact with the adenosine phosphate of FAD . However, several prokaryotic E3s have Val instead of Thr . To investigate the meaning and importance of the Thr conservation in many E3s, the corresponding residue, Thr-44, in human E3 was substituted to Val by site-directed mutagenesis . The mutant's E3 activity showed about a 2.2-fold decrease . Its UV-visible and fluorescence spectra indicated that the mutant might have a slightly different microenvironment at the FAD-binding region. Environ Med, 1999 Dec, 43(2), 79 - 87 The role of heat shock proteins in mammalian differentiation and development; Walsh D et al.; Heat shock proteins (HSPs) have been identified in all cells, prokaryotic and eukaryotic, to protect the cells from harmful insults and stress . Increased HSP synthesis can also result during normal cellular functions and also respond to exposure from environmental stress and infection . Although the molecular mechanisms responsible for HSP cellular protection are still not fully understood, their expression is critical for cellular survival and can be modified by cell signal transducers such as intracellular pH, cyclic AMP, Ca2+ Na+, inositol {correction of inostitol} triphosphate, protein kinase C, and protein phosphates . Most HSPs interact, assemble, fold, unfold, bind, transport, translocate and 'chaperone' other proteins in the cell and alter their function. J Bacteriol, 2002 Oct, 184(20), 5733 - 45 Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures; Ma J et al.; This work assesses relationships for 30 complete prokaryotic genomes between the presence of the Shine-Dalgarno (SD) sequence and other gene features, including expression levels, type of start codon, and distance between successive genes . A significant positive correlation of the presence of an SD sequence and the predicted expression level of a gene based on codon usage biases was ascertained, such that predicted highly expressed genes are more likely to possess a strong SD sequence than average genes . Genes with AUG start codons are more likely than genes with other start codons, GUG or UUG, to possess an SD sequence . Genes in close proximity to upstream genes on the same coding strand in most genomes are significantly higher in SD presence . In light of these results, we discuss the role of the SD sequence in translation initiation and its relationship with predicted gene expression levels and with operon structure in both bacterial and archaeal genomes. J Bacteriol, 2002 Oct, 184(20), 5641 - 53 Adaptive, or stationary-phase, mutagenesis, a component of bacterial differentiation in Bacillus subtilis; Sung HM et al.; Adaptive (stationary-phase) mutagenesis occurs in the gram-positive bacterium Bacillus subtilis . Furthermore, taking advantage of B . subtilis as a paradigm for the study of prokaryotic differentiation and development, we have shown that this type of mutagenesis is subject to regulation involving at least two of the genes that are involved in the regulation of post-exponential phase prokaryotic differentiation, i.e., comA and comK . On the other hand, a functional RecA protein was not required for this type of mutagenesis . The results seem to suggest that a small subpopulation(s) of the culture is involved in adaptive mutagenesis and that this subpopulation(s) is hypermutable . The existence of such a hypermutable subpopulation(s) raises important considerations with respect to evolution, the development of specific mutations, the nature of bacterial populations, and the level of communication among bacteria in an ecological niche. J Bacteriol, 2002 Oct, 184(20), 5554 - 62 Distinct clpP genes control specific adaptive responses in Bacillus thuringiensis; Fedhila S et al.; ClpP and ClpC are subunits of the Clp ATP-dependent protease, which is ubiquitous among prokaryotic and eukaryotic organisms . The role of these proteins in stress tolerance, stationary-phase adaptive responses, and virulence in many bacterial species has been demonstrated . Based on the amino acid sequences of the Bacillus subtilis clpC and clpP genes, we identified one clpC gene and two clpP genes (designated clpP1 and clpP2) in Bacillus thuringiensis . Predicted proteins ClpP1 and ClpP2 have approximately 88 and 67% amino acid sequence identity with ClpP of B . subtilis, respectively . Inactivation of clpC in B . thuringiensis impaired sporulation efficiency . The clpP1 and clpP2 mutants were both slightly susceptible to salt stress, whereas disruption of clpP2 negatively affected sporulation and abolished motility . Virulence of the clp mutants was assessed by injecting bacteria into the hemocoel of Bombyx mori larvae . The clpP1 mutant displayed attenuated virulence, which appeared to be related to its inability to grow at low temperature (25 degrees C), suggesting an essential role for ClpP1 in tolerance of low temperature . Microscopic examination of clpP1 mutant cells grown at 25 degrees C showed altered bacterial division, with cells remaining attached after septum formation . Analysis of lacZ transcriptional fusions showed that clpP1 was expressed at 25 and 37 degrees C during the entire growth cycle . In contrast, clpP2 was expressed at 37 degrees C but not at 25 degrees C, suggesting that ClpP2 cannot compensate for the absence of ClpP1 in the clpP1 mutant cells at low temperature . Our study demonstrates that ClpP1 and ClpP2 control distinct cellular regulatory pathways in B . thuringiensis. Biochemistry, 2002 Oct 1, 41(39), 11750 - 60 Regulation of actin dynamics by tyrosine phosphorylation: identification of tyrosine phosphorylation sites within the actin-severing domain of villin; Zhai L et al.; We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation . Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly . We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T . Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene . Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261) . Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256 . Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay . Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin . Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin . Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function. Biochemistry, 2002 Oct 1, 41(39), 11703 - 10 Structural and energetic analysis of metal ions essential to SRP signal recognition domain assembly; Batey RT et al.; The signal recognition particle (SRP) targets proteins to the endoplasmic reticulum in eukaryotes or to the inner membrane in prokaryotes by binding to hydrophobic signal sequences . Signal peptide recognition occurs within the highly conserved RNA-protein core of the SRP, underscoring the importance of this complex in SRP function . Structural analysis of the RNA and protein components of the prokaryotic SRP in the free and bound states revealed that the RNA undergoes a significant conformational change upon protein binding involving the uptake of several monovalent and divalent cations . To investigate the role of these metal ions in formation of the functional SRP complex, we used binding affinity assays and X-ray crystallography to analyze the specificity and energetic contributions of mono- and divalent metal ions bound in the RNA . Our results demonstrate that several metal ion binding sites important for RNA conformation can accommodate chemically distinct ions, often without affecting the structure of the complex . Thus, while these metal ions are highly ordered and essential for the formation and stability of the SRP complex, they behave like nonspecific metal ions. Poult Sci, 2002 Sep, 81(9), 1317 - 21 Stimulatory effect of CpG sequences on humoral response in chickens; Vleugels B et al.; Oligonucleotides containing the dinucleotide CpG have an immunostimulatory effect in mammals . The CpG motif is interpreted as a signal of prokaryote invasion by the innate immune system and consequently activates defense mechanisms . The goal of this study was to investigate whether the immunostimulatory actions of CpG oligonucleotides take place in birds as well . To this end, birds were immunized with BSA and the serum antibody response was followed . A significantly higher BSA-specific response was observed in the CpG-treated group . Moreover, immunostimulatory DNA resulted in more persistent responses to immunization . After only one immunization, titers as high as in booster responses were observed in the CpG-treated birds . The effects were shown to depend on the presence of an unmethylated CpG core sequence in the DNA, because the reversal of CpG to GpC even caused a decrease in antibody response . These findings demonstrate that CpG oligonucleotides could be a valuable adjuvant for poultry vaccines. Genetics, 2002 Sep, 162(1), 217 - 27 Identification and analysis of a hyperactive mutant form of Drosophila P-element transposase; Beall EL et al.; Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions . However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein . In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation . In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type . This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert . The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI(3)-related protein kinases . Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo . A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions . These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition. Biochem J, 2002 Oct 1, 367(Pt 1), 219 - 27 Cloning and characterization of anti-cathepsin L single chain variable fragment whose expression inhibits procathepsin L secretion in human melanoma cells; Guillaume-Rousselet N et al.; We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic . Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv . For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems . An anti-cathepsin L monoclonal antibody (mAb), named 3D8, was isolated from mice immunized with purified procathepsin L-His . This 3D8 mAb interacted with an epitope localized on the 156-197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells . An active anti-cathepsin L ScFv was generated and characterized from 3D8 mAb heavy and light variable chains . Then, human melanoma cells were transiently co-transfected with 3D8 ScFv and cathepsin L cDNAs . Data demonstrated that increase of 3D8 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L . Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion . This ScFv represents a new molecular tool to explore cell therapy of human melanomas. Mol Biol Rep, 2002, 29(1-2), 227 - 32 Virtual mitochondria: metabolic modelling and control; Aimar-Beurton M et al.; Inside the eukaryotic cell, mitochondria are internal organelles of prokaryotic origin, responsible for energy supply in the cell . The control of the mitochondrial ATP production is a complex problem with different patterns according to different tissues and organs . Our aim is to continue to develop the modelling of oxidative phosphorylation in different tissues, to model other parts of mitochondrial metabolism and to include this virtual mitochondria in a virtual cell . In constructing the complete metabolic map of mitochondria, we will take advantage of the sequenced genomes of eukaryotic organism (10-15% of the yeast genome concerns mitochondria). Biotechniques, 2002 Sep, 33(3), 672, 674, 676 - 8 Development of multiple cloning site cis-vectors for recombinant adeno-associated virus production; Yue Y et al.; Recombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years . A cis-plasmid is used to generate the rAAV stocks . In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats . The construction of cis-plasmid has been problematic because of the high-frequency recombination of the viral inverted terminal repeats . Here we describe the design and construction of several multiple cloning site cis-plasmids that are driven by five different promoters, including the ubiquitous cytomegalovirus enhancer/chicken beta-actin (CAG), cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus 40 (SV40), and a muscle-specific promoter (CK6) . The application of these multiple cloning site cis-plasmids improves the cloning efficiency . As an example of the utilization of these multiple cloning site vectors, the prokaryotic beta-galactosidase cDNA was cloned in the multiple cloning site cis-plasmids . High-level rAAV-mediated beta-galactosidase expression was achieved in HeLa cells from CAG, CMV, RSV and SV40 promoters, respectively, but notfrom the CK6 promoter . In vivo application in the adult mdx mouse (mouse model for Duchenne muscular dystrophy) muscle revealed efficient transgene expression from CMV and CK6 promoters, followed by CAG and RSV promoters . The SV40 promoter was the least efficient. Biotechniques, 2002 Sep, 33(3), 548 - 50, 552, 554-6 GWFASTA: server for FASTA search in eukaryotic and microbial genomes; Issac B et al.; Similarity searches are a powerful method for solving important biological problems such as database scanning, evolutionary studies, gene prediction, and protein structure prediction . FASTA is a widely used sequence comparison tool for rapid database scanning . Here we describe the GWFASTA server that was developed to assist the FASTA user in similarity searches against partially and/or completely sequenced genomes . GWFASTA consists of more than 60 microbial genomes, eight eukaryote genomes, and proteomes of annotatedgenomes . Infact, it provides the maximum number of databases for similarity searching from a single platform . GWFASTA allows the submission of more than one sequence as a single query for a FASTA search . It also provides integrated post-processing of FASTA output, including compositional analysis of proteins, multiple sequences alignment, and phylogenetic analysis . Furthermore, it summarizes the search results organism-wise for prokaryotes and chromosome-wise for eukaryotes . Thus, the integration of different tools for sequence analyses makes GWFASTA a powerful toolfor biologists. Southeast Asian J Trop Med Public Health, 2002 Jun, 33(2), 288 - 96 Recombinant envelope protein of HIV-1 subtype E as antigen in HIV-1 antibody detection enzyme immunoassay; Sutthent R et al.; In order to develop a reliable and inexpensive serodiagnostic method to be used for anti-HIV antibody detection in Thailand, recombinant envelope (TM or gp41 subunit) protein of HIV-1 subtype E was produced from prokaryotic cell (Escherichia coli) as the source of antigen in enzyme immunoassay (TE diagnostic EIA kit) . HIV-1 gp41 subunit of subtype E was successfully expressed in E . coli in the form of polyhistidine-tagged proteins, comprising of rgp41A (601 bases N-terminal half of TM or 25kDa) and rgp41B (560 bases C-terminal half of TM or 24 kDa) by using an expression vector, pBAD/His C . The amount of protein, dilution of sera, and anti-human IgG labeled HRP used in the EIA test optimized by a checker board titration of the protein and seropositive or seronegative sera, were 5.0 microg/ml, 1:300, and 1:4,000, respectively . The blinded test evaluation of TE-diagnostic EIA in 500 seropositive and 500 seronegative sera which have been simultaneously tested by two available commercial kits and compared with our TE diagnostic EIA, gave 99.6% sensitivity and specificity . The other known genetic subtypes sera such as subtype A (n=5), B (n=9), C (n=4) and D (n=5) were also positive with this EIA . The estimated manufacturer cost per test of rgp41 based anti-HIV antibody detection EIA or TE-diagnostic EIA was about 15 baht . This recombinant envelope (gp41 or TM) protein from HIV-1, which can be produced in large quantities without any hazards from growing the virus and has lower cost to produce anti-HIV antibody serological diagnostic kit, should be considered as an HIV screening test in Thailand. FASEB J, 2002 Oct, 16(12), 1639 - 41 Epub 2002 Aug 07. The alpha-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor myogenin; Corbi N et al.; RNA polymerase II core subunit 3 (RPB3) is an a-like core subunit of RNA polymerase II (pol II) . It is selectively down-regulated upon treatment with doxorubicin (dox) . Due to the failure of skeletal muscle cells to differentiate when exposed to dox, we hypothesized that RPB3 is involved in muscle differentiation . To this end, we have isolated human muscle RPB3-interacting proteins by using yeast two-hybrid screening . It is of interest that an interaction between RPB3 and the myogenic transcription factor myogenin was identified . This interaction involves a specific region of RPB3 protein that is not homologous to the prokaryotic a subunit . Although RPB3 contacts the basic helix-loop-helix (HLH) region of myogenin, it does not bind other HLH myogenic factors such as MyoD, Myf5, and MRF4 . Coimmunoprecipitation experiments indicate that myogenin contacts the pol II complex and that the RPB3 subunit is responsible for this interaction . We show that RPB3 expression is regulated during muscle differentiation . Exogenous expression of RPB3 slightly promotes myogenin transactivation activity and muscle differentiation, whereas the region of RPB3 that contacts myogenin, when used as a dominant negative molecule (Sud), counteracts these effects . These results indicate for the first time that the RPB3 pol II subunit is involved in the regulation of tissue-specific transcription. J Theor Biol, 2002 Aug 21, 217(4), 479 - 92 A study of the middle-scale nucleotide clustering in DNA sequences of various origin and functionality, by means of a method based on a modified standard deviation; Nikolaou C et al.; The deviation from randomness in the distribution of nucleotides in genomic sequences is quantified and studied, using a modified standard deviation (MSD) . This method implies a "per block" computation of the standard deviation of the nucleotide frequencies of occurrence, using local means (means taken in a neighborhood of each block) . This quantity may serve as a scale-dependent measure of the nucleotide clustering . In the present work, the meso-scale of tenths of nucleotides is principally explored, by means of suitably adjusted filter parameters . This length scale is of an order of magnitude not directly affected by the grammar and syntax rules of the protein-coding procedure, remaining shorter than the scale of appearance of large-scale characteristics of the genome . MSD has been found to distinguish systematically between the sequences of different origin and functionality . The most near-random are found to be coding sequences of prokaryotes, while in intronic and intergenic regions of eukaryotic genomes, extended clustering of similar nucleotides is observed . The distributions of MSD values of large collections of sequences are found to be in most cases characteristic of their biological role and origin . Protein- and non-coding, prokaryotic and eukaryotic DNA as well as promoter, rRNA, viral and organelle sequences have been examined . The presented results corroborate a recently proposed model for genome evolution . The method is also applied for an assessment of the annotation of ORFs taken from the complete genome of Saccharomyces cerevisiae. Curr Microbiol, 2002 Nov, 45(5), 355 - 61 A conserved Ala320 in the FtsZ of Porphyromonas gingivalis is important for cell division; Yu W et al.; We have previously cloned the gene encoding the cell division protein FtsZ, designated PgFtsZ, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease . In the present study, we have shown that overexpression of ZDeltaC02, a mutant form of PgFtsZ in which 128 amino acid residues have been removed from the C-terminus, caused an inhibition of cell division in E . coli cells . However, overexpression of ZDeltaC03, missing 177 residues from the C-terminus, did not inhibit cell division, suggesting that the 49 residues between 281 and 329 are required for cell division . Sequence comparison of the known prokaryotic FtsZs revealed that this region contained a highly conserved domain, designated A-domain, in which Ala320 of PgFtsZ was conserved throughout a broad variety of species . Therefore, we analyzed the role of Ala320 by site-directed mutagenesis . We found that overexpression of ZA320H and ZA320R resulted in the normal phenotype, unlike the wild type . Immunoblot analysis showed that these mutant proteins were expressed at similar levels . These results suggest that Ala320 is highly conserved and is crucial for cell division. Plant Physiol, 1994 Sep, 106(1), 143 - 150 A Mutant of Arabidopsis Deficient in the Elongation of Palmitic Acid; Wu J et al.; The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by an increased level of 16:0 and a concomitant decrease of 18-carbon fatty acids as a consequence of a single recessive nuclear mutation at the fab1 locus . Quantitative analysis of the fatty acid composition of individual lipids established that lipids synthesized by both the prokaryotic and eukaryotic pathways were affected by the mutation . Direct enzyme assays demonstrated that the mutant plants were deficient in the activity of 3-ketoacyl-acyl carrier protein synthase II; therefore, it is inferred that fab1 may encode this enzyme . Labeling experiments with {14C}acetate and lipase positional analysis indicated that the mutation results in a small shift in the partitioning of lipid synthesis between the prokaryotic and eukaryotic pathways . Synthesis of chloroplast lipids by the prokaryotic pathway was increased with a corresponding reduction in the eukaryotic pathway. Plant Physiol, 1993 Nov, 103(3), 733 - 739 Purification and Properties of a Monofunctional Imidazoleglycerol-Phosphate Dehydratase from Wheat; Mano J et al.; Imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19) activity was detected in extracts of several monocotyledonous and dicotyledonous plants using a newly developed assay method . The enzyme was purified 114,000-fold (to apparent homogeneity) from wheat germ by five chromatographic steps . Its native relative molecular weight (Mr) was determined to be 600,000 to 670,000, and it consists of identical subunits of Mr 25,500 . In wheat germ, the dehydratase, unlike those of prokaryotic origin, is not associated with histidinol phosphatase activity . The reaction product was identified as imidazoleacetol phosphate (IAP) by comparing it with synthetic IAP as an authentic reference . The Km value for imidazoleglycerol phosphate was 0.36 mM at the optimal pH of 6.6 . The enzyme required a reducing agent, such as 2-mercaptoethanol or dithiothreitol, and Mn2+ for maximal activity . 3-Amino-1,2,4-triazole competitively inhibited the activity with a Ki value of 46 {mu}M . The purification of imidazoleglycerol-phosphate dehydratase from wheat germ and histidinol dehydrogenase from cabbage (A . Nagai, A . Scheidegger {1991} Arch Biochem Biophys 284: 127-132) suggests that at least the second half of the histidine biosynthesis in plants is identical to that in microorganisms. Acta Pol Pharm, 2002 May-Jun, 59(3), 199 - 207 Synthesis of 6-substituted 6H-indolo{2,3-b}quinolines as novel cytotoxic agents and topoisomerase II inhibitors; Kaczmarek L et al.; A systematic investigation into the impact of the substituents introduced into the indolo{2,3-b}quinoline system is described . The findings clearly demonstrate that the compounds bearing a methyl group or a longer aliphatic chain at the N-6 position are inactive against prokaryotic and eukaryotic cells . The introduction of alkyl-amino-alkyl substituent at the N-6 position of indolo{2,3-b}quinoline accounts for the appearance of the antimicrobial and.cytotoxic properties . The cytotoxicity against oral epidermoid carcinoma KB (ID50) is in the range from 2.0 to 9.0 microM, and the antimicrobial activity (MIC) falls between 0.03 and 0.50 mM . The structural relation within 6H-indolo{2,3-b}quinolines, concerning their antimicrobial and cytotoxic activity, corresponds well with their ability to bind DNA and to inhibit topoisomerase II activity. Infect Immun, 2002 Oct, 70(10), 5827 - 34 Isolation of Chlamydia pneumoniae clonal variants by a focus-forming assay; Gieffers J et al.; Chlamydia pneumoniae is an obligate intracellular prokaryotic human pathogen that causes community-acquired respiratory infection and has been associated with atherosclerosis and cardiovascular disease . Unexpected results from genomic sequencing indicate that significant intrastrain polymorphism exists for some C . pneumoniae isolates . These polymorphisms could reflect genotypes with differing disease-causing characteristics . A definitive means to test this hypothesis is to obtain genetically homogeneous clonal populations of the pathogen and test them in models of infection and disease . To date, methods for cloning C . pneumoniae have not been reported . In this study, we describe the isolation of clonal variants with genetic differences in the tyrP locus from a polymorphic respiratory isolate, using a novel focus-forming assay . These results now allow investigations on the biology and pathogenesis of C . pneumoniae clonal genovars that could lead to new insights into the pathogenesis of this important human pathogen. J Biol Chem, 2002 Dec 6, 277(49), 47325 - 30 Epub 2002 Sep 11. X-ray structure of two crystalline forms of a streptomycete ribonuclease with cytotoxic activity; Sevcik J et al.; Ribonuclease (RNase) Sa3 is secreted by the Gram-positive bacterium Streptomyces aureofaciens . The enzyme catalyzes the cleavage of RNA on the 3' side of guanosine residues . Here, x-ray diffraction analysis was used to determine the three-dimensional structure of two distinct crystalline forms of RNase Sa3 to a resolution of 2.0 and 1.7 A . These two structures are similar to each other as well as to that of a homolog, RNase Sa . All of the key active-site residues of RNase Sa (Asn(42), Glu(44), Glu(57), Arg(72), and His(88)) are located in the putative active site of RNase Sa3 . Also herein, RNase Sa3 is shown to be toxic to human erythroleukemia cells in culture . Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein . Thus, a prokaryotic ribonuclease can be toxic to mammalian cells. Curr Infect Dis Rep, 2002 Oct, 4(5), 387 - 399 The Pathophysiology and Treatment of Candida Sepsis; Spellberg B et al.; Sepsis can occur during disseminated candidiasis, but its pathogenesis differs from that caused by typical prokaryotic pathogens . Complex interactions between defects in host defense and "relative" virulence factors expressed by Candida lead to dissemination of the saprophyte to parenchymal organs, and subsequently to onset of multiorgan failure . This review focuses first on the pathophysiology of Candida sepsis, detailing current understanding of host-pathogen interactions . We then consider the choice of antifungal and supportive treatments. Plant Physiol, 2002 Sep, 130(1), 477 - 86 Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression; Lu S et al.; Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species . A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases . The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (-)-beta-pinene and (-)-alpha-pinene in a 94:6 ratio . QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting . QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments . Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase) . The contents of (-)-beta-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation . When A . annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms . Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed . However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod . This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern. J Biol Chem, 2002 Nov 29, 277(48), 46304 - 9 Epub 2002 Sep 10. Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain; Sandowski Y et al.; A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein . The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins . The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m . Similar results were achieved with conventional binding experiments . LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor . The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex . Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding. Prog Biophys Mol Biol, 2002 May-Jul, 79(1-3), 77 - 133 Molecular evolution before the origin of species; Davis BK; Amino acids at conserved sites in the residue sequence of 10 ancient proteins, from 844 phylogenetically diverse sources, were used to specify their time of origin in the interval before species divergence from the last common ancestor (LCA) . The order of amino acid addition to the genetic code, based on biosynthesis path length and other molecular evidence, provided a reference for evaluating the 'code age' of each residue profile examined . Significantly earlier estimates were obtained for conserved amino acid residues in these proteins than non-conserved residues . Evidence from the primary structure of 'fossil' proteins thus corroborated the biosynthetic order of amino acid addition to the code.Low potential ferredoxin (Fdxn) had the earliest residue profile among the proteins in this study . A phylogenetic tree for 82 prokaryote Fdxn sequences was rooted midway between bacteria and archaea branches . LCA Fdxn had a 23-residue antecedent whose residue profile matched mid-expansion phase codon assignments and included an amide residue . It contained a highly acidic N-terminal region and a non-charged C-terminal region, with all four cysteine residues . This small protein apparently anchored a {4Fe-4S} cluster, ligated by C-terminal cysteines, to a positively charged mineral surface, consistent with mediating e(-) transfer in a primordial surface system before cells appeared . Its negatively charged N-terminal 'attachment site' was highly mutable during evolution of ancestral Fdxn for Bacteria and Archaea, consistent with a loss of function after cell formation . An initial glutamate to lysine substitution may link 'attachment site' removal to early post-expansion phase entry of basic amino acids to the code . As proteins evidently anchored non-charged amide residues initially, surface attachment of cofactors and other functional groups emerges as a general function of pre-cell proteins.A phylogenetic tree of 107 proteolipid (PL) helix-1 sequences from H(+)-ATPase of bacteria, archaea and eukaryotes had its root between prokaryote branches . LCA PL h1 residue profile optimally fit a late expansion phase codon array . Sequence repeats in transmembrane PL helices h1 and h2 indicated formation of the archetypal PL hairpin structure involved successive tandem duplications, initiated within the gene for an 11-residue (or 4-residue) hydrophobic peptide . Ancestral PL h1 lacked acidic residues, in a fundamental departure from the prototype pre-cell protein . By this stage, proteins with a hydrophobic domain had evolved . Its non-polar, late expansion phase residue profile point to ancestral PL being a component of an early permeable cell membrane . Other indicators of cell formation about this stage of code evolution include phospholipid biosynthesis path length, FtsZ residue profile, and late entry of basic amino acids into the genetic code.Estimates based on conserved residues in prokaryote cell septation protein, FtsZ, and proteins involved with synthesis, transcription and replication of DNA revealed FtsZ, ribonucleotide reductase, RNA polymerase core subunits and 5'-->3' flap exonuclease, FEN-1, originated soon after cells putatively evolved . While reverse transcriptase and topoisomerase I, Topo I, appeared late in the pre-divergence era, when the genetic code was essentially complete . The transition from RNA genes to a DNA genome seemingly proceeded via formation of a DNA-RNA heteroduplex . These results suggest formation of DNA awaited evolution of a catalyst with a hydrophobic domain, capable of sequestering radical bearing intermediates in its synthesis from ribonucleotide precursors . Late formation of topology altering protein, Topo I, further suggests consolidation of genes into chromosomes followed synthesis of comparatively thermostable DNA strands. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2002 Jun, 19(2), 280 - 3 {Expression and purification of human papillomavirus type16 L1 protein in a prokaryotic expression system}; Bao G et al.; This study was intended to establish a method of purification of HPV16 L1 protein expressed in a prokaryotic system and to obtain the purified protein . The prokaryotic expression vector pGEX-4T-1-HPV16 L1 was constructed and transformed into E . coli BL21 cell, and induced by 1 mM IPTG to express HPV16L1 protein . The inclusion bodies were isolated and solubilized with 8 M urea . After the urea was removed by gradual dialysis, the denatured L1 protein were renatured and then were purified by affinity chromatography . The results showed that HPV16L1 protein formed inclusion bodies in bacterial expression system, suggesting that this assay can be used to purify HPV16L1 protein and hence provide a basis for studying the applications of HPV16 L1 protein. Plant Physiol, 1997 May, 114(1), 223 - 230 Elucidation of the Biosynthesis of Eicosapentaenoic Acid in the Microalga Porphyridium cruentum (II . Studies with Radiolabeled Precursors); Khozin I et al.; In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors . Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature . The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid . However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, {omega}6 and {omega}3, which involve cytoplasmic and chloroplastic lipids . In the {omega}6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4{omega}6-PC by a sequence that includes a {delta}6 desaturase, an elongation step, and a {delta}5 desaturase . In the minor {omega}3 pathway, 18:2-PC is presumably desaturated to 18:3{omega}3, which is sequentially converted by the enzymatic sequence of the {omega}6 pathway to 20:5{omega}3-PC . The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species . The 20:4{omega}6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic {delta}17 ({omega}3) desaturase. Cell Mol Life Sci, 2002 Jul, 59(7), 1081 - 95 The galactosyltransferase family; Hennet T; Galactose is transferred via several linkages to acceptor structures by galactosyltransferase enzymes . In prokaryotes, galactose is mainly found on lipopolysaccharides and capsular polysaccharides . In eukaryotes, galactosyltransferases, which are localized in the Golgi apparatus, are involved in the formation of several classes of glycoconjugates and in lactose biosynthesis . Although they sometimes catalyze identical reactions, prokaryotic and eukaryotic galactosyltransferases share only little structural similarities . In mammals, 19 distinct galactosyltransferase enzymes have been characterized to date . These enzymes catalyze the transfer of galactose via beta1-4, beta1-3, alpha1-3 and alpha1-4 linkages . The present review focuses on the description of these mammalian galactosyltransferases. Trends Biotechnol, 2002 Oct, 20(10), 407 - 10; discussion 410 Conservation of gene co-regulation in prokaryotes and eukaryotes; Teichmann SA et al.; Genes that are part of the same operon in prokaryotes, or have the same expression pattern in eukaryotes, are transcriptionally co-regulated . If genes are consistently co-regulated across distantly related organisms, the genes have closely associated functions . It has been shown previously that such genes have a strong tendency to belong to the same protein complex in prokaryotes, and we show by an analysis of the sequences and their expression in the yeast Saccharomyces cerevisiae and the worm Caenorhabditis elegans that this is also true for eukaryotes . Our analysis reveals that the number of conserved co-regulated genes is small in eukaryotes, as has been shown previously in prokaryotes, indicating that there are extensive variations in the gene regulatory network across organisms. FEBS Lett, 2002 Sep 11, 527(1-3), 319 - 22 Posttranslational modification of E . coli histone-like protein H-NS and bovine histones by short-chain poly-(R)-3-hydroxybutyrate (cPHB); Reusch RN et al.; Short-chain poly-(R)-3-hydroxybutyrate (cPHB), a highly flexible, amphiphilic molecule with salt-solvating properties, is a ubiquitous constituent of prokaryotic and eukaryotic cells, wherein it is mainly conjugated to proteins . The solvating properties and cellular distribution of cPHB suggest it may be associated with proteins that bind and/or transfer DNA . Here we examine Escherichia coli protein H-NS and calf thymus histones, H1, H2A, H2B, H3, and H4, for the presence of cPHB . The proteins are related in that all bind to DNA and are implicated in the compact organization of the chromosome . The presence of cPHB in E . coli H-NS was first detected in Western blots of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of total cell proteins, probed with anti-cPHB IgG, and then by Western blot analysis of the purified protein . Western blot analysis of the calf thymus histones indicated that each contained cPHB . The presence of cPHB in H-NS and histones was confirmed by chemical assay . The in vivo size of conjugated cPHB could not be established due to the lack of standards and degradation of cPHB during protein purification and storage . The molecular characteristics of cPHB and its presence in histone-like and histone proteins of diverse organisms suggest it may play a role in DNA binding and/or DNA organization. Biochem Biophys Res Commun, 2002 Sep 13, 297(1), 10 - 6 Functional properties of Kch, a prokaryotic homologue of eukaryotic potassium channels; Munsey TS et al.; To test the hypothesis that the Kch gene of Escherichia coli encodes a potassium channel, we have transformed E . coli with an expression vector containing the Kch sequence and observed the effect of over-expression of Kch on E . coli . We found that: (i) over-expression of Kch is toxic to E . coli, but the toxicity could be prevented by supplementing the growth medium with K(+), Rb(+), and NH(4)(+), but not Na(+), consistent with the properties of a potassium selective pore; (ii) Cs(+), a blocker of potassium channels, rescues the growth of Kch over-expressing cells; and (iii) when the putative pore-forming region of Kch, containing the signature sequence, was replaced with the corresponding region of the eukaryotic Shaker potassium channel, and the resultant construct expressed in E . coli, the cells became critically dependent on K(+) supply for survival . These data are consistent with the proposed function of Kch, i.e., K(+) conduction. Environ Microbiol, 2002 Sep, 4(9), 546 - 55 Extremely productive microbial communities in shallow saline pools respond immediately to changing meteorological conditions; Kirschner AK et al.; Diel changes in bacterial and cyanobacterial numbers, as well as heterotrophic bacterial production, were examined in two shallow alkaline pools, harbouring dense populations of cyanobacteria (up to 1100 x 109 cells l-1) and bacteria (up to 500 x 109 cells l-1) . Together with the recorded bacterial production rates (925 micro gC l-1x h-1), these values are the highest reported for natural aquatic ecosystems . The investigations were performed during a fair-weather situation, and during a rapid change after a long-term fair-weather situation to thunderstorms and heavy rainfall . During fair weather, bacterial growth was significantly correlated to the diurnal light and temperature cycle . Prokaryotic abundances were fairly constant, and loss by grazing and viral lysis must have been of significant importance . During the invasion of rainy weather, the prokaryotic community showed a strong and immediate response . A significant enhancement of bacterial growth followed after rainfall, suggesting that the high salt concentrations had inhibited bacterial activity . Changes in bacterial and cyanobacterial numbers were consistent with this pattern . From comparison with the available literature, we conclude that diel changes of bacterioplankton are regulated by a complex combination of environmental factors specific for each investigated ecosystem . In the soda pools investigated, external abiotic factors were dominant on a diel scale . In larger ecosystems, such factors are much more buffered and internal biotic interactions may prevail. Biochemistry, 2002 Sep 17, 41(37), 11071 - 9 Structure of a nitric oxide synthase heme protein from Bacillus subtilis; Pant K et al.; Eukaryotic nitric oxide synthases (NOSs) produce nitric oxide to mediate intercellular signaling and protect against pathogens . Recently, proteins homologous to mammalian NOS oxygenase domains have been found in prokaryotes and one from Bacillus subtilis (bsNOS) has been demonstrated to produce nitric oxide {Adak, S., Aulak, K . S., and Stuehr, D . J . (2002) J . Biol . Chem . 277, 16167-16171} . We present structures of bsNOS complexed with the active cofactor tetrahydrofolate and the substrate L-arginine (L-Arg) or the intermediate N(omega)-hydroxy-L-arginine (NHA) to 1.9 or 2.2 A resolution, respectively . The bsNOS structure is similar to those of the mammalian NOS oxygenase domains (mNOS(ox)) except for the absence of an N-terminal beta-hairpin hook and zinc-binding region that interact with pterin and stabilize the mNOS(ox) dimer . Changes in patterns of residue conservation between bacterial and mammalian NOSs correlate to different binding modes for pterin side chains . Residue conservation on a surface patch surrounding an exposed heme edge indicates a likely interaction site for reductase proteins in all NOSs . The heme pockets of bsNOS and mNOS(ox) recognize L-Arg and NHA similarly, although a change from Val to Ile beside the substrate guanidinium may explain the 10-20-fold slower dissociation of product NO from the bacterial enzyme . Overall, these structures suggest that bsNOS functions naturally to produce nitrogen oxides from L-Arg and NHA in a pterin-dependent manner, but that the regulation and purpose of NO production by NOS may be quite different in B . subtilis than in mammals. J Mol Biol, 2002 Aug 30, 321(5), 785 - 96 Crystal structure and anion binding in the prokaryotic hydrogenase maturation factor HypF acylphosphatase-like domain; Rosano C et al.; {NiFe}-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes . One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone . The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster . It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases . The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates . The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong . Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains . The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families . On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain . The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain. Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12015 - 20 Epub 2002 Sep 06. Physical constraints and functional characteristics of transcription factor-DNA interaction; Gerland U et al.; We study theoretical "design principles" for transcription factor (TF)-DNA interaction in bacteria, focusing particularly on the statistical interaction of the TFs with the genomic background (i.e., the genome without the target sites) . We introduce and motivate the concept of programmability, i.e., the ability to set the threshold concentration for TF binding over a wide range merely by mutating the binding sequence of a target site . This functional demand, together with physical constraints arising from the thermodynamics and kinetics of TF-DNA interaction, leads us to a narrow range of "optimal" interaction parameters . We find that this parameter set agrees well with experimental data for the interaction parameters of a few exemplary prokaryotic TFs, which indicates that TF-DNA interaction is indeed programmable . We suggest further experiments to test whether this is a general feature for a large class of TFs. Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12246 - 51 Epub 2002 Sep 06. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus; Martin W et al.; Chloroplasts were once free-living cyanobacteria that became endosymbionts, but the genomes of contemporary plastids encode only approximately 5-10% as many genes as those of their free-living cousins, indicating that many genes were either lost from plastids or transferred to the nucleus during the course of plant evolution . Previous estimates have suggested that between 800 and perhaps as many as 2,000 genes in the Arabidopsis genome might come from cyanobacteria, but genome-wide phylogenetic surveys that could provide direct estimates of this number are lacking . We compared 24,990 proteins encoded in the Arabidopsis genome to the proteins from three cyanobacterial genomes, 16 other prokaryotic reference genomes, and yeast . Of 9,368 Arabidopsis proteins sufficiently conserved for primary sequence comparison, 866 detected homologues only among cyanobacteria and 834 other branched with cyanobacterial homologues in phylogenetic trees . Extrapolating from these conserved proteins to the whole genome, the data suggest that approximately 4,500 of Arabidopsis protein-coding genes ( approximately 18% of the total) were acquired from the cyanobacterial ancestor of plastids . These proteins encompass all functional classes, and the majority of them are targeted to cell compartments other than the chloroplast . Analysis of 15 sequenced chloroplast genomes revealed 117 nuclear-encoded proteins that are also still present in at least one chloroplast genome . A phylogeny of chloroplast genomes inferred from 41 proteins and 8,303 amino acids sites indicates that at least two independent secondary endosymbiotic events have occurred involving red algae and that amino acid composition bias in chloroplast proteins strongly affects plastid genome phylogeny. Bioinformatics, 2002 Sep, 18(9), 1266 - 7 SEGE: A database on 'intron less/single exonic' genes from eukaryotes; Sakharkar MK et al.; SUMMARY: Eukaryotes have both 'intron containing' and 'intron less' genes . Several databases are available for 'intron containing' genes in eukaryotes . In this note, we describe a database for 'intron less' genes from eukaryotes . 'Intron less' eukaryotic genes having prokaryotic architecture will help to understand gene evolution in a much simpler way unlike 'intron containing' genes . AVAILABILITY: SEGE is available at CONTACT: mmeena@ntu.edu.sg Bioinformatics, 2002 Sep, 18(9), 1153 - 61 Genome structures embossed by oligonucleotide-stickiness; Nishigaki K et al.; MOTIVATION: An unmanageably large amount of data on genome sequences is accumulating, prompting researchers to develop new methods to analyze them . We have devised a novel method designated oligostickiness, a measure roughly proportional to the binding affinity of an oligonucleotide to a DNA of interest, in order to analyze genome sequences as a whole . RESULTS: Fifteen representative genomes such as Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, H . sapiens and others were analyzed by this method using more than 50 probe dodecanucleotides, offering the following findings: (i) Genome sequences can be specifically featured by way of oligostickiness maps . (ii) Oligostickiness analysis, which is similar to but more informative than (G + C) content or repetitive sequence analysis, can reveal intra-genomic structures such as mosaic structures (E . coli and B . subtilis) and highly sticky/non-sticky regions of biological meanings . (iii) Some probe oligonucleotides such as dC(12) and dT(12) can be used for classifying genomes, clearly discriminating prokaryotes and eukaryotes . (iv) Based on global oligostickiness, which is the average value of the local oligostickinesses, the features of a genome could be visualized in spider web mode . The pattern of a spider web as well as a set of oligostickiness maps is highly characteristic to each genome or chromosome . Thus, we called it as chromosome texture, leading to a finding that all the chromosomes contained in a cell, so far investigated, have a common texture . AVAILABILITY: Oligostickinesses maps used in this work are available at CONTACT: koichi@fms.saitama-u.ac.jp Extremophiles, 2002 Aug, 6(4), 275 - 81 Epub 2002 Mar 08. L-aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: characteristics and role in the de novo biosynthesis of nicotinamide adenine dinucleotide proposed by genome sequencing; Sakuraba H et al.; A gene encoding the L-aspartate oxidase homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3 . We succeeded in expressing the encoding gene in Escherichia coli and purified the product to homogeneity . Characterization of the protein revealed that it is the most thermostable L-aspartate oxidase detected so far . In addition to the oxidase activity, the enzyme catalyzed L-aspartate dehydrogenation in the presence of an artificial electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, and ferricyanide . L-Aspartate oxidase is known to function as the first enzyme in the de novo NAD biosynthetic pathway in prokaryotes . By a similarity search in public databases, the genes that encode the homologue of all other enzymes involved in the pathway were identified in the P . horikoshii OT-3 genome . This suggests that P . horikoshii OT-3 may use the de novo NAD biosynthetic pathway under anaerobic conditions. Science, 2002 Sep 6, 297(5587), 1686 - 9 Identification of a DNA nonhomologous end-joining complex in bacteria; Weller GR et al.; In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ) . Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes . The NHEJ pathway requires a DNA end-binding component called Ku . We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer . Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation . Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis . These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged. J Biol Chem, 2002 Nov 22, 277(47), 45075 - 85 Epub 2002 Sep 04. Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework; der Maur AA et al.; Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells . These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins . However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form . In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies . We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection . Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies. J Mol Biol, 2002 Sep 6, 322(1), 53 - 64 Loopy proteins appear conserved in evolution; Liu J et al.; Over the last decade, structural biologists have unravelled many proteins that appear natively disordered . Common assumptions are that many of these proteins adopt structure through binding and that the structural flexibility enables them to adopt different functions . Here, we investigated regions of more than 70 sequence-consecutive residues that have no regular secondary structure (NORS) . Analysing 31 entirely sequenced organisms, we predicted five times as many proteins with NORS regions (loopy proteins) in eukaryotes (20%) than in prokaryotes and archaeas (4%) . Thousands of these NORS regions were over 150 residues long . The amino acid composition of NORS regions differed from that of loops in PDB . Although NORS proteins had significantly more residues in low-complexity regions than other proteins, simple cut-off thresholds for sequence bias missed most NORS regions . On average, NORS regions were evolutionarily at least as conserved as their flanking regions . Furthermore, yeast proteins with NORS regions had more protein-protein interaction partners than other proteins . Regulatory and transcription-related functions were over-represented in loopy proteins, biosynthesis and energy metabolism were under-represented . Overall, our analysis confirmed that proteins with non-regular structures appear to play important functional roles, and they may adopt as yet unknown types of protein structures. Biochim Biophys Acta, 2002 Sep 13, 1577(2), 325 - 36 Regulation of viral transcription elongation and termination during vaccinia virus infection; Condit RC et al.; Vaccinia virus provides a useful genetic and biochemical tool for studies of the basic mechanisms of eukaryotic transcription . Vaccinia genes are transcribed in three successive gene classes during infection, early, intermediate, and late . Vaccinia transcription is regulated primarily by virus gene products not only during initiation, but also during elongation and termination . The factors and mechanisms regulating early elongation and termination differ from those regulating intermediate and late gene expression . Control of transcription elongation and termination in vaccinia virus bears some similarity to the same process in other prokaryotic and eukaryotic systems, yet features some novel mechanisms as well. Biochim Biophys Acta, 2002 Sep 13, 1577(2), 287 - 307 Promoting elongation with transcript cleavage stimulatory factors; Fish RN et al.; Transcript elongation by RNA polymerase is a dynamic process, capable of responding to a number of intrinsic and extrinsic signals . A number of elongation factors have been identified that enhance the rate or efficiency of transcription . One such class of factors facilitates RNA polymerase transcription through blocks to elongation by stimulating the polymerase to cleave the nascent RNA transcript within the elongation complex . These cleavage factors are represented by the Gre factors from prokaryotes, and TFIIS and TFIIS-like factors found in archaea and eukaryotes . High-resolution structures of RNA polymerases and the cleavage factors in conjunction with biochemical investigations and genetic analyses have provided insights into the mechanism of action of these elongation factors . However, there are yet many unanswered questions regarding the regulation of these factors and their effects on target genes. Biochim Biophys Acta, 2002 Sep 13, 1577(2), 191 - 207 Promoter clearance and escape in prokaryotes; Hsu LM; Promoter escape is the last stage of transcription initiation when RNA polymerase, having initiated de novo phosphodiester bond synthesis, must begin to relinquish its hold on promoter DNA and advance to downstream regions (DSRs) of the template . In vitro, this process is marked by the release of high levels of abortive transcripts at most promoters, reflecting the high instability of initial transcribing complexes (ITCs) and indicative of the existence of barriers to the escape process . The high abortive initiation level is the result of the existence of unproductive ITCs that carry out repeated initiation and abortive release without escaping the promoter . The formation of unproductive ITCs is a widespread phenomenon, but it occurs to different extent on different promoters . Quantitative analysis of promoter mutations suggests that the extent and pattern of abortive initiation and promoter escape is determined by the sequence of promoter elements, both in the promoter recognition region (PRR) and the initial transcribed sequence (ITS) . A general correlation has been found that the stronger the promoter DNA-polymerase interaction, the poorer the ability of RNA polymerase to escape the promoter . In gene regulation, promoter escape can be the rate-limiting step for transcription initiation . An increasing number of regulatory proteins are known to exert their control at this step . Examples are discussed with an emphasis on the diverse mechanisms involved . At the molecular level, the X-ray crystal structures of RNA polymerase and its various transcription complexes provide the framework for understanding the functional data on abortive initiation and promoter escape . Based on structural and biochemical evidence, a mechanism for abortive initiation and promoter escape is described. Bioessays, 2002 Sep, 24(9), 850 - 7 The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene; Spinelli G et al.; Going back to the late 1970s and early 1980s, we trace the Xenopus oocyte microinjection experiments that led to the emergence of the concept of "modulator" . The finding that the modulator could transactivate transcription from far upstream and in either orientation suggested that a new genetic element, different from the classical prokaryotic promoter sequences, had been discovered . This particular enhancer transactivates transcription of the sea urchin early (alpha) histone H2A gene which is regulated in early sea urchin development . We summarise the data from sea urchin microinjection experiments that confirm and extend the results obtained with Xenopus oocytes . We conclude that the H2A enhancer is bipartite, is located approx . 100 bp upstream of the TATAAATA box in the H2A gene of two sea urchin species and enhances transcription when placed at a position far upstream or far downstream of the gene unless an insulator intervenes between enhancer and promoter . Evidence from microinjection experiments with sea urchin embryos suggests that the developmental control of H2A expression resides not with the enhancer, which is constitutively active, but with a striking chromatin structure with two positioned nucleosomes near the 3' end of the gene . Within this structure, there is an insulator element . Biotechnol Bioeng, 2002 Sep 5, 79(5), 504 - 8 A memorial review of Jay Bailey's contribution in prokaryotic metabolic engineering; Hatzimanikatis V et al.; When mentioning prokaryotic metabolic engineering, most people will immediately think of Jay Bailey . Jay's contribution to this fast-growing field is evident and familiar to many . Therefore, instead of a detailed technical review, we attempt in this article to summarize his contribution and dissect reasons for his success in this area from a standpoint of one of his former students (VH) and of a colleague in the field (JCL) . This short review is by no means complete and provides only a partial view of Jay's contribution to the metabolic engineering of prokaryotes . Mol Microbiol, 2002 Sep, 45(5), 1365 - 77 The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome; Jakimowicz D et al.; The mycelial prokaryote Streptomyces coelicolor A3(2) possesses a large linear chromosome (8.67 Mb) with a centrally located origin of replication (oriC) . Recently, chromosome partitioning genes (parA and parB) and putative ParB binding sites (parS sequences) were identified in its genome . The S . coelicolor chromosome contains more parS sequences than any other bacterial chromosome characterized so far . Twenty of the 24 parS sequences are densely packed within a relatively short distance (approximately 200 kb) around oriC . A series of in vitro and in vivo experiments showed that S . coelicolor ParB protein interacts specifically with the parS sequences, albeit with a rather low affinity . Our results suggested that the binding of ParB is not only determined by the parS sequence, but also by the location of target DNA close to oriC . The unusually high number and close proximity to each other of the parS sites, together with in vivo and in vitro evidence that multiple ParB molecules may assemble along the DNA from an initial ParB-parS complex, suggest that a large DNA segment around the replication origin may form a massive nucleoprotein complex as part of the replication-partitioning cycle. Mol Microbiol, 2002 Sep, 45(5), 1191 - 6 The dynamic microbe: green fluorescent protein brings bacteria to light; Southward CM et al.; The demonstration that the green fluorescent protein (GFP) from the jellyfish Aequorea victoria required no jellyfish-specific cofactors and could be expressed as a fluorescent protein in heterologous hosts including both prokaryotes and eukaryotes sparked the development of GFP as one of the most common reporters in use today . Over the past several years, the utility of GFP as a reporter has been optimized through the isolation and engineering of variants with increased folding rates, different in vivo stabilities and colour variants with altered excitation and emission spectral properties . One of the great utilities of GFP is as a probe for characterizing spatial and temporal dynamics of gene expression, protein localization and protein-protein interactions in living cells . The innovative application of GFP as a reporter in bacteria has made a significant contribution to microbial cell biology . This review will highlight recent studies that demonstrate the potential of GFP for real-time analysis of gene expression, protein localization and the dynamics of signalling transduction pathways through protein-protein interactions. Nat Biotechnol, 2002 Sep, 20(9), 901 - 7 Epub 2002 Aug 19. Macrolide-based transgene control in mammalian cells and mice; Weber W et al.; Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing . The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (E(OFF)) and inducible (E(ON)) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin) . The E(OFF) system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (P(ETR)) . Addition of macrolide antibiotic results in repression of transgene expression . The E(ON) system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression . In the presence of macrolides, gene expression is induced . Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (E(OFF) and E(ON)) systems . The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP-regulated and tetracycline (TET-regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues. Biosystems, 2002 Jun-Jul, 66(1-2), 65 - 71 Changes of the power coefficient in the 'metabolism-mass' relationship in the evolutionary process of animals; Atanasov AT et al.; The power coefficient k decreases along evolution in an allometric relationship between the oxygen consumption rate and the body mass of animals . This theoretical study investigated the role of the power coefficient k and its behavior along evolution . The animals were organized in three groups according to the values of the power coefficient k as follows: (I) from unicellular Prokaryotes to Eukaryotes; (II) from Mytilus and Annelida to Pisces; (III) from Reptilia to Mammals and Aves . At the beginning of each animal group (stage), the value of k was close to 0.9-1.0 and at the end of the stage it was close to 0.67-0.70 . Exponential sharp increase of the power coefficient k was observed during the biological transition from Protozoa to simply organized Metazoa and in the transition from Poikylothermic to Homothermic organisms (e.g . from Pisces to Reptilia) . Also, when using the periodogram regression analysis, a cyclic (periodic) pattern in this increase was observed (i.e . period T approximately 8-11 units, P<0.05) . It was postulated that the power coefficient k, as with the coefficient a, might represent the increase of complexity of animal organization within each group. Int J Parasitol, 2002 Sep, 32(10), 1207 - 17 Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists; Seeber F; During the last 4 years there has been an enormous interest in the question how iron-sulphur ({Fe-S}) clusters, which are essential building blocks for life, are synthesised and assembled into apo-proteins, both in prokaryotes and in eukaryotes . The emerging picture is that the basic mechanism of this pathway has been well conserved during evolution . In yeast and probably all other eukaryotes the mitochondrion is the place where {Fe-S} clusters are synthesised, even for extramitochondrial {Fe-S} cluster-containing proteins, and a number of proteins have been functionally characterised to a certain extent within this pathway . However, almost nothing is known about this aspect in parasitic protists, although recent studies of amitochondriate protists and on the plastid-like organelle of apicomplexan parasites, the apicoplast, have started to change this . In this article I will summarise the current view of {Fe-S} cluster biogenesis in eukaryotes and discuss its implications for amitochondriate protists and for the plastid-like organelle of apicomplexan parasites. J Biochem (Tokyo), 2002 Sep, 132(3), 433 - 9 Spectroscopic analysis of recombinant rat histidine decarboxylase; Olmo MT et al.; Mammalian histidine decarboxylases have not been characterized well owing to their low amounts in tissues and instability . We describe here the first spectroscopic characterization of a mammalian histidine decarboxylase, i.e . a recombinant version of the rat enzyme purified from transformed Escherichia coli cultures, with similar kinetic constants to those reported for mammalian histidine decarboxylases purified from native sources . We analyzed the absorption, fluorescence and circular dichroism spectra of the enzyme and its complexes with the substrate and substrate analogues . The pyridoxal-5'-phosphate-enzyme internal Schiff base is mainly in an enolimine tautomeric form, suggesting an apolar environment around the coenzyme . Michaelis complex formation leads to a polarized, ketoenamine form of the Schiff base . After transaldimination, the coenzyme-substrate Schiff base exists mainly as an unprotonated aldimine, like that observed for dopa decarboxylase . However, the coenzyme-substrate Schiff base suffers greater torsion than that observed in other L-amino acid decarboxylases, which may explain the relatively low catalytic efficiency of this enzyme . The active center is more resistant to the formation of substituted aldamines than the prokaryotic homologous enzyme and other L-amino acid decarboxylases . Characterization of the similarities and differences of mammalian histidine decarboxylase with respect to other homologous enzymes would open new perspectives for the development of new and more specific inhibitors with pharmacological potential. Environ Toxicol, 2002, 17(4), 341 - 50 Genotoxicity of cyanobacterial extracts containing microcystins from Polish water reservoirs as determined by SOS chromotest and comet assay; Mankiewicz J et al.; Toxicity of cyanobacterial blooms, an increasing problem around the world, is connected to the increase in bloom samples containing microcystins, caused by excessive eutrophication of drinking- and recreational water reservoirs . Microcystins are the most common group of cyanobacterial hepatotoxins . In Poland they are produced mainly by the Microcystis genus . The toxicity of microcystins has been well documented, but investigation into their genotoxicity has been insufficient relative to the study of their overall toxicity . Therefore, the aim of this study was the estimation and comparison of the genotoxicity of cyanobacterial extracts with microcystins (CEMs) using the SOS chromotest (bacterial test) with Escherichia coli PQ37 and the comet assay with human lymphocytes . Cyanobacterial bloom samples were collected in the summer months from two Polish water reservoirs, one at Sulejow and one at Jeziorsko . The SOS chromotest, which used prokaryotic cells (without metabolic activation), and the comet assay, which used eukaryotic cells, both indicated the potential genotoxic effect of CEMs . Cyanobacterial extracts caused DNA damage in human lymphocytes in vitro . The maximum level of DNA damage was observed after 12 h incubation with CEMs . The bacterial test indicated a dependence of the degree of CEM genotoxicity, the composition, and the concentration of microcystins in each bloom sample examined with the time of exposure . Differences between the genotoxicity of cyanobacterial extract and the standard microcystin-LR were noticeable . This was probably caused by the interaction of different microcystin variants . The results showed that CEMs from Polish water reservoirs were genotoxic, which was reflected by the stimulation of the SOS repair system in bacterial cells (SOS chromotest) and by the damage induced in DNA in human lymphocytes (comet assay) . Nucleic Acids Res, 2002 Sep 1, 30(17), 3643 - 52 Novel domains and orthologues of eukaryotic transcription elongation factors; Ponting CP; The passage of RNA polymerase II across eukaryotic genes is impeded by the nucleosome, an octamer of histones H2A, H2B, H3 and H4 dimers . More than a dozen factors in the yeast Saccharomyces cerevisiae are known to facilitate transcription elongation through chromatin . In order to better understand the evolution and function of these factors, their sequences have been compared with known protein, EST and DNA sequences . Elongator subcomplex components Elp4p and Elp6p are shown to be homologues of ATPases, yet with substitutions of amino acids critical for ATP hydrolysis, and novel orthologues of Elp5p are detectable in human, and other animal, sequences . The yeast CP complex is shown to contain a likely inactive homologue of M24 family metalloproteases in Spt16p/Cdc68p and a 2-fold repeat in Pob3p, the orthologue of mammalian SSRP1 . Archaeal DNA-directed RNA polymerase subunit E" is shown to be the orthologue of eukaryotic Spt4p, and Spt5p and prokaryotic NusG are shown to contain a novel 'NGN' domain . Spt6p is found to contain a domain homologous to the YqgF family of RNases, although this domain may also lack catalytic activity . These findings imply that much of the transcription elongation machinery of eukaryotes has been acquired subsequent to their divergence from prokaryotes. Cell, 2002 Aug 23, 110(4), 407 - 13 Shared strategies in gene organization among prokaryotes and eukaryotes; Lawrence JG; Although genes in prokaryotes and eukaryotes are transcribed and translated by very different mechanisms, they may be organized in their respective chromosomes in surprisingly similar ways . Here, I examine common modes of maintaining nonrandom gene organization in both prokaryotes and eukaryotes, the different ways these organizations have likely arisen, and classes of organization that may be unique to one group or the other. Curr Opin Genet Dev, 2002 Oct, 12(5), 512 - 8 Intramembrane proteolysis controls diverse signalling pathways throughout evolution; Urban S et al.; Regulated intramembrane proteolysis (RIP) has recently emerged as a conserved mechanism for controlling many signalling pathways in both prokaryotes and eukaryotes . Although early examples were confined to the activation of membrane-tethered transcription factors in the cell receiving the signal, recent analysis indicates that RIP also regulates the emission of factors involved in intercellular communication. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 675 - 9 {Expression, refolding and biological activity of recombinant type-I metalloproteinase acutolysin a from Agkistrodon acutus}; Xiang KJ et al.; Type-I snake venom metalloproteinase acutolysin A gene was cloned into the prokaryotic expression vector pBAD/gIIIA and the resulting recombinant plasmid pDS was obtained . By the induction with 0.02% L-(+)-arabinose, the recombinant metalloproteinase was expressed in insoluble inclusion body in E . coli TOP10 and reached up to 5%--10% of total bacterial proteins . The recombinant metalloproteinase has an additional sequence of N-terminal 22 amino acids and C-terminal 8 amino acids (housing 6 histidines), both of which derived from the vector . The purified inclusion body was solubilized by 8 mol/L urea or 6 mol/L guanidine-HCl and the denatured soluble recombinant metalloproteinase was allowed to refold in vitro . Western blotting and ELISA obviously showed that the renatured recombinant metalloproteinase possessed strong immune reactivity very closely related to natural acutolysin A . Animal experiments showed that the refolded recombinant metalloproteinase had an obvious hemorrhagic activity . Except PMSF, 1 mmol/L EDTA, 1 mmol/L EGTA, and 3 mmol/L imidazole could inhibit the hemorrhagic activity of the recombinant and the natural metalloproteinases to different extent . Based on the investigations of others and our experimental results, the hemorrhagic mechanism of snake metalloproteinases was discussed. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 671 - 4 {Preparation of polyacrylamide gel electrophoresis for human chorionic gonadotropin chimeric peptide 12 expressed in E . coli}; Zou YS et al.; In recent years, development of chimeric peptide (CP) immunogens is a trend in the vaccinological field . The CPs contain a B cell epitope(s) of target antigen and a promiscuous self - or foreign- T cell epitope(s) . However, such constructed CPs were all expressed in prokaryotic or eukaryotic systems at lower levels . To purify the human chorionic gonadotropin (hCG) CP12 expressed in E . coli at the level of about 1% of the total cell proteins, an improved method of preparative gel polyacrylamide gel electrophoresis (PAGE) was developed . The important improvement to routine preparative PAGE involves: (1) running reversed electrophoresis by rearranging the gel- carrying plate when the bromophenol blue band arrived at 1-1.5 centimeter from the bottom of the gel; (2) making a collecting trough between the gel and a dialytic membrane that was used to isolate the upper tank buffer . About 8 fractions were collected at regular intervals of 15 minutes after bromophenol blue running out of gel . And then 0.2 ml was taken from each fraction and the protein was precipitated by sequentially adding trichloroacetic acid and acetone . Each sample was dissolved in 20 microL sample buffer and analyzed and identified by SDS-PAGE and Western blotting . As a result, the hCG CP12 expression product with 95% relative homogeneity was harvested at a 50-100 microgram level after a single-step purification of this preparative PAGE, with respect to the sample which contained 3-4 mg of cell proteins. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 595 - 600 {Expression, purification and characterization of human prorelaxin-like-protein H2 in Escherichia coli}; Chen LM et al.; The cDNA gene encoding human prorelaxin H2 was subcloned from plasmid pMAL p2X-hRLX H2 into the EcoRI and BamHI sites of prokaryotic expression vector pBV220, resulting in the recombinant plasmid pBV220-hPRLX H2, which was subsequently transformed into Escherichia coli DH5arg; . The target gene was highly expressed in the form of inclusion body by thermoinduction at 42 degrees . Recombinant human Met-prorelaxin-like-protein H2 was refolded in vitro, purified by Sephadex G-75 gel filtration chromatography and reverse phase fast protein liquid chromatography . The yield was approximately 2-3 mg/L . The purified recombinant human Met-prorelaxin-like-protein H2 was shown to be a single band by 15% SDS-PAGE and gave correct amino acid composition of Met-prorelaxin . Molecular weight of the purified recombinant human Met-prorelaxin-like-protein H2 was measured to be 18 390.4 (calculated theoretical value 18 392.3) by matrix assisted laser desorption ionization time-of-flight mass spectroscopy. Nature, 2002 Aug 29, 418(6901), 942 - 8 Open channel structure of MscL and the gating mechanism of mechanosensitive channels; Perozo E et al.; Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations . This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation . Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling . MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology . Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1) . Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions . The open state is highly dynamic, supporting a water-filled pore of at least 25 A, lined mostly by TM1 . These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels. J Biol Chem, 2002 Nov 1, 277(44), 41401 - 9 Epub 2002 Aug 26. Ribosomal proteins at the stalk region modulate functional rRNA structures in the GTPase center; Uchiumi T et al.; Replacement of the L10.L7/L12 protein complex and L11 in Escherichia coli ribosomes with the respective rat counterparts P0.P1/P2 and eukaryotic L12 causes conversion of ribosomal specificity for elongation factors from prokaryotic elongation factor (EF)-Tu/EF-G to eukaryotic EF (eEF)-1alpha/eEF-2 . Here we have investigated the effects of protein replacement on the structure and function of two rRNA domains around positions 1070 and 2660 (sarcin/ricin loop) of 23 S rRNA . Protein replacement at the 1070 region in E . coli 50 S subunits was demonstrated by chemical probing analysis . Binding of rat proteins to the 1070 region caused increased accessibility of the 2660 and 1070 regions to ligands for eukaryotic ribosomes: the ribotoxin pepocin for the 2660 region (E . coli numbering), anti-28 S autoantibody for the 1070 region, and eEF-2 for both regions . Moreover, binding of the E . coli L10.L7/L12 complex and L11 to the 1070 region was shown to be responsible for E . coli ribosomal accessibility to another ribotoxin, gypsophilin . Ribosomal proteins at the 1070 region appear to modulate the structures and functions of the 2660 and 1070 RNA regions in slightly different modes in prokaryotes and eukaryotes. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 184 - 6 {Expression, purification and diagnostic application of EBV TK kinase}; Huang B et al.; BACKGROUND: To find a rapid and sensitive method for early diagnosis of nasopharyngeal carcinoma by using EBV TK kinase . METHODS: Prokaryotic expression plasmid pRSETTK was constructed . EBV TK kinase was highly expressed in E.coil BL21 (DE3) . The authors identified specificity of TK kinase by Western blot, then used purified TK kinase in ELISA to detect the IgG antibody in the serum of NPC patients . RESULTS: Specific IgG antibody against TK kinase was found in the serum of NPC patients . The specificity and sensitivity of TK kinase were both 100% in Western blot and were 98.0% and 93.4% respectively in ELISA . CONCLUSIONS: The EBV TK kinase showed high specificity and sensitivity in ELISA, therefore it can be used for early diagnosis of NPC Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 176 - 8 {Cloning and expression of HSV-I, II type-common antigen gD in Escherichia coli}; Li M et al.; BACKGROUND: To clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD . METHODS: The authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha . After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA . RESULTS: The constructed expression vector pMAL-c2/gD can be expressed with high efficiency . The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm . CONCLUSIONS: The authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6 . Therefore the protein was of natural antigenic structure of gD. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 168 - 70 {Cloning and expression of simplex herpes virus ? US4 fragment}; Yi Y et al.; BACKGROUND: To get early laboratory study of type specific antigenicity of herpes simplex virus II . METHODS: PCR and prokaryotic expression technique . RESULTS: Herpes simplex virus II type specific gene fragment was expressed in E.coli and the products can be used as specific antigen for the detection of anti\HSV in the recovery sera . CONCLUSIONS: Cloning and express of HSVII type specific antigen found the basis for developing specific diagnosis methods and vaccine of HSV. Biochem Soc Trans, 2002 Aug, 30(4), 397 - 401 14-3-3s are DNA-replication proteins; Zannis-Hadjopoulos M et al.; 14-3-3 proteins are conserved multifunctional molecules, involved in many biological processes . Several 14-3-3 isoforms were recently shown to be cruciform DNA-binding proteins, which is a new activity ascribed to the 14-3-3 family . As cruciform-binding proteins, 14-3-3 proteins are putatively involved in the regulation of DNA replication . Inverted repeat sequences that are able to extrude into cruciform structures are a common feature of replication origins in both prokaryotes and eukaryotes . The involvement of cruciform structures in the initiation of DNA replication has been demonstrated . A leading model of 14-3-3 function proposes that they facilitate critical protein-protein interactions, thus serving as a central component of a wide variety of cellular processes. Postepy Hig Med Dosw, 2002, 56(3), 263 - 71 {Molecular basis of ion selectivity based on crystalline structure of bacterial channels}; Koprowski P et al.; The principles of ionic selectivity of the two crystallised bacterial ion channels are described . These channels are: the potassium channel KcsA, whose amino-acid sequence is homologous to the eukaryotic voltage--dependent potassium channels and the chloride channel EcClC that is a prokaryotic member of the ClC family of chloride channels . In conclusion: although the overall molecular architecture of KcsA is different from that of EcClC, the selectivity filters in both cases show similarities . They both utilise helix dipoles organised within the channel molecule in such a fashion to produce electrostatically favourable environment for anions (in the case of EcClC) or cations (in the case of KcsA). Ann Endocrinol (Paris), 2002 Jun, 63(3), 197 - 218 {Water homeostasis in the living: molecular organization, osmoregulatory reflexes and evolution}; Acher R; Human body weight is about 70% water; 55% of the water are in cells and 45% in extracellular compartments, mainly body fluids . Each compartment has its own osmoregulatory system . The mechanisms of intracellular osmoregulation likely appeared with the cell itself, i.e . in primitive prokaryotes, some 3.8 billions years ago . Osmotic stress responses observed in present-day bacteria, yeast, plant and animals cells in culture are very similar . Variations in the cell volume entail an extension or retraction of plasma membrane that activates mechanically-gated ion channels or mechanoreceptors . Some of them have been cloned from E . coli, the nematode C . elegans, the drosophila . Osmotic stress determines an intracellular cascade of transactivations, the last transcription factor binding to a Tonicity response element (TonE) of osmoprotective genes . These genes encode enzymes synthesizing compatible osmolytes that reequilibrate the osmotic pressure . Volume and osmolality of the biological fluids (le milieu interieur) are regulated by neuroendocrine reflexes involving an afferent neural limb from baro- and osmo-receptors to hypothalamus and an efferent endocrine limb from neurosecretory cells to target cells, the hydroosmotic cells localized in osmoregulatory organs . Afferent signals trigger the biosynthesis and the processing of neurohypophyseal preprohormones in magnocellular neurons of the supraoptic and paraventricular nuclei of hypothalamus . Vasopressin in mammais and vasotocin in other vertebrates, endowed with antidiuretic and antinatriuretic properties, act on hydroosmotic cells localized in the nephron collecting duct . A specific vasopressin receptor, the V2 type receptor, located in the basolateral membrane of the principal cells, is coupled with an heterotridimeric protein Gs that activates adenylate cyclase . The cAMP product, in turn, stimulates the protein kinase A (PKA) . The latter mobilizes 5 effectors located in the apical membrane: 1) the water channel aquaporin 2; 2) the urea transporter UT1; 3) the amiloride-sensitive sodium channel ENaC; 4) the inward-rectifying potassium channel ROM-K1; 5) the chloride channel CFTR . Modulation of each effector is ensured by an A kinase anchoring protein (AKAP) . The effectors are transported from cytosol to apical membrane through transport vesicles equipped with membrane fusion proteins (such as VAMP2) that recognize specific apical membrane proteins (such as syntaxins) . Water flows into principal cells from collecting duct lumen through AQP2 and flows out to interstitium through AQP3 and AQP4 and finally reachs blood circulation through AQP1 capillaries . Vasopressin orchestrates the live effectors so as to keep water and solutes reabsorptions in balance with volaemia and osmolality set points. J Bacteriol, 2002 Sep, 184(18), 5104 - 12 Transposition of cyanobacterium insertion element ISY100 in Escherichia coli; Urasaki A et al.; The genome of the cyanobacterium Synechocystis sp . strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant . A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences . It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family . To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid . Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency . Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements . Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence . No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have . These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision . Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision . No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system . ISY100 transposes in E . coli, indicating that it transposes without any host factor other than the transposase encoded by itself . Therefore, it may be able to transpose in other biological systems. Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 331 - 4 {Study on construct and expression of synthetic genes encoding spider dragline silk in Escherichia coli}; Li M et al.; Dragline spider silk produced from Nephilia clavipes major ampullate is a natural fibrous protein with specific mechanical properties such as high tensile strength and elasticity . Synthetic gene monomer encoding recombinant spider silk protein, based on the known repetitive protein sequence and partial cDNA sequence of dragline silk, was constructed and expressed . DNA monomer sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy . Multimer was cloned into pET30a(+), a prokaryotic high potency expression vector, and induced with IPTG . The protein from 8-unit repeat was produced in Escherichia coli at levels up to 20 mg/L . The protein was easily purified with high recovery by using an metal ion affinity chromatography and purity was over 90% . The results of SDS-PAGE and Western blot suggested that the mass of the expression product was about 37 kD . This value and amino acid analysis were consistent with those of theoretic calculation. Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 318 - 22 {Study on optimization of expression, purification, properties and biological function of recombinant human sBLyS}; Yan XM et al.; The prokaryotic expression plasmid pET-30a(+)/sBLyS was constructed and transformed into E . coli BL21 (lambda DE3) . The recombinant protein was found to be highly expressed by the plight of soluble part and inclusion body . For the sake of enhancing the proportion of the soluble part, inducement at 16 degrees C for 12 h was ascertained . The expressing product was then purified by Ni2+ affinity chromatography gel . PI of the recombinant human sBLyS(rhsBLyS) is about 7.1-7.3 and it assembles into a homotrimer . The effect of rhsBLyS on B lymphocytes by MTT method told us the B lymphocytes' proliferating capacity dose depended on concentration and also stimulating time of the rhsBLys . With rhsBLyS(2 micrograms/mL) stimulating 3 days, B lymphocytes can proliferate the most. Protein Sci, 2002 Sep, 11(9), 2196 - 207 The beta-barrel finder (BBF) program, allowing identification of outer membrane beta-barrel proteins encoded within prokaryotic genomes; Zhai Y et al.; Many outer membrane proteins (OMPs) in Gram-negative bacteria possess known beta-barrel three-dimensional (3D) structures . These proteins, including channel-forming transmembrane porins, are diverse in sequence but exhibit common structural features . We here report computational analyses of six outer membrane proteins of known 3D structures with respect to (1) secondary structure, (2) hydropathy, and (3) amphipathicity . Using these characteristics, as well as the presence of an N-terminal targeting sequence, a program was developed allowing prediction of integral membrane beta-barrel proteins encoded within any completely sequenced prokaryotic genome . This program, termed the beta-barrel finder (BBF) program, was used to analyze the proteins encoded within the Escherichia coli genome . Out of 4290 sequences examined, 118 (2.8%) were retrieved . Of these, almost all known outer membrane proteins with established beta-barrel structures as well as many probable outer membrane proteins were identified . This program should be useful for predicting the occurrence of outer membrane proteins in bacteria with completely sequenced genomes. Biochim Biophys Acta, 2002 Sep 2, 1592(1), 79 - 87 Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex; Stuart R; The inner mitochondrial membrane harbors a large number of proteins that display a wide range of topological arrangements . The majority of these proteins are encoded in the cell's nucleus, but a few polytopic proteins, all subunits of respiratory chain complexes are encoded by the mitochondrial genome . A number of distinct sorting mechanisms exist to direct these proteins into the mitochondrial inner membrane . One of these pathways involves the export of proteins from the matrix into the inner membrane and is used by both proteins synthesized within the mitochondria, as well as by a subset of nuclear encoded proteins . Prior to embarking on the export pathway, nuclear encoded proteins using this sorting route are initially imported into the mitochondrial matrix from the cytosol, their site of synthesis . Protein export from the matrix into the inner membrane bears similarities to Sec-independent protein export in bacteria and requires the function of the Oxa1 protein . Oxa1 is a component of a general protein insertion site in yeast mitochondrial inner membrane used by both nuclear and mitochondrial DNA encoded proteins . Oxa1 is a member of the conserved Oxa1/YidC/Alb3 protein family found throughout prokaryotes throughout eukaryotes (where it is found in mitochondria and chloroplasts) . The evidence to demonstrate that the Oxa1/YidC/Alb3 protein family represents a novel evolutionarily conserved membrane insertion machinery is reviewed here. Mol Cell, 2002 Aug, 10(2), 409 - 15 Structure of the yeast RNA polymerase II holoenzyme: Mediator conformation and polymerase interaction; Davis JA et al.; The holoenzyme formed by RNA polymerase II (RNAPII) and the Mediator complex is the target of transcriptional regulators in vivo . A three-dimensional structure of the yeast holoenzyme has been generated from electron microscopic images of single holoenzyme particles . Extensive changes in Mediator conformation required for interaction with RNAPII have been modeled by correlating the polymerase-bound and free Mediator structures . Determination of the precise orientation of the RNAPII in the holoenzyme indicates that Mediator contacts are centered on the RNAPII Rpb3/Rpb11 heterodimer, the eukaryotic homolog of the alpha(2) homodimer involved in transcription regulation in prokaryotes . Implications for the possible mechanism of transcription regulation by Mediator are discussed. BMC Genomics . 2002 Aug 21;3(1):26. Mouse ribonuclease III . cDNA structure, expression analysis, and chromosomal location; Fortin KR et al.; BACKGROUND: Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse RNA maturation and decay pathways in eukaryotic and prokaryotic cells . A human RNase III orthologue has been implicated in ribosomal RNA maturation . To better understand the structure and mechanism of mammalian RNase III and its involvement in RNA metabolism we determined the cDNA structure, chromosomal location, and expression patterns of mouse RNase III . RESULTS: The predicted mouse RNase III polypeptide contains 1373 amino acids (approximately 160 kDa) . The polypeptide exhibits a single C-terminal dsRNA-binding motif (dsRBM), tandem catalytic domains, a proline-rich region (PRR) and an RS domain . Northern analysis and RT-PCR reveal that the transcript (4487 nt) is expressed in all tissues examined, including extraembryonic tissues and the midgestation embryo . Northern analysis indicates the presence of an additional, shorter form of the transcript in testicular tissue . Fluorescent in situ hybridization demonstrates that the mouse RNase III gene maps to chromosome 15, region B, and that the human RNase III gene maps to a syntenic location on chromosome 5p13-p14 . CONCLUSIONS: The broad transcript expression pattern indicates a conserved cellular role(s) for mouse RNase III . The putative polypeptide is highly similar to human RNase III (99% amino acid sequence identity for the two catalytic domains and dsRBM), but is distinct from other eukaryotic orthologues, including Dicer, which is involved in RNA interference . The mouse RNase III gene has a chromosomal location distinct from the Dicer gene. Mikrobiol Z, 2002 Mar-Apr, 64(2), 82 - 94 {Fructose-bisphosphatase of microorganisms}; Skrypal' IG et al.; Data from modern scientific literature concerning general characteristics of fructose-bisphosphatase--the key enzyme of microorganisms gluconeogenesis pathway have been analyzed in the survey . The conditions of fructose-bisphosphatase functioning in the cell--activation and repression, have been described . Regulation of prokaryotes enzyme activity by fructose-1.6-bisphosphate, fructose-2.6-bisphosphate, phosphoenol pyruvate, metal cation, etc., is discussed . Absolute dependence of fructose-bisphosphatase activity on cations of bivalent metals (Mg2+, Mn2+) and AMP is shown . Special attention was given to the functioning and characteristics of the enzyme in prokaryotes and eukaryotes: their similarity and differences . Data about the basic enzyme of gluconeogenesis pathway in Gram-positive and Gram-negative microorganisms, conditions of its functioning in autotrophs and heterotrophs have been generalized for the first time . Genetical and molecular biological properties of fructose-bisphosphatase in microorganisms have been considered . At the same time, the fact is established that the above enzyme has not been investigated in numerous microorganisms. EMBO Rep, 2002 Sep, 3(9), 881 - 6 Epub 2002 Aug 16. Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1; Seit-Nebi A et al.; In eukaryotic ribosomes, termination of translation is triggered by class 1 polypeptide release factor, eRF1 . In organisms with a universal code, eRF1 responds to three stop codons, whereas, in ciliates with variant codes, only one or two codon(s) remain(s) as stop signals . By mutagenesis of the Y-C-F minidomain of the N domain, we converted an omnipotent human eRF1 recognizing all three stop codons into a unipotent 'ciliate-like' UGA-only eRF1 . The conserved Cys127 located in the Y-C-F minidomain plays a critical role in stop codon recognition . The UGA-only response has also been achieved by concomitant substitutions of four other amino acids located at the Y-C-F and NIKS minidomains of eRF1 . We suggest that for eRF1 the stop codon decoding is of a non-linear (non-protein-anticodon) type and explores a combination of positive and negative determinants . We assume that stop codon recognition is profoundly different by eukaryotic and prokaryotic class 1 RFs. J Mol Evol, 2002 Sep, 55(3), 260 - 4 Aerobiosis increases the genomic guanine plus cytosine content (GC%) in prokaryotes; Naya H et al.; The huge variation in the genomic guanine plus cytosine content (GC%) among prokaryotes has been explained by two mutually exclusive hypotheses, namely, selectionist and neutralist . The former proposals have in common the assumption that this feature is a form of adaptation to some ecological or physiological condition . On the other hand, the neutralist interpretation states that the variations are due only to different mutational biases . Since all of the traits that have been proposed by the selectionists either appeared to be limited to certain genera or were invalidated by the availability of more data, they cannot be considered as a selective force influencing the genomic GC% across all prokaryotes . In this report we show that aerobic prokaryotes display a significant increment in genomic GC% in relation to anaerobic ones . This is the first time that a link between a metabolic character and GC% has been found, independently of phylogenetic relationships and with a statistically significant amount of data. J Biochem Mol Biol Biophys, 2002 Apr, 6(2), 117 - 21 Cloning and expression of the phosphoprotein gene of Newcastle disease virus in Escherichia coli; Kho CL et al.; The phosphoprotein (P) gene of a heat stable Newcastle disease virus (NDV) was cloned, sequenced and expressed in Escherichia coli . SDS-PAGE analysis of the recombinant P protein (395 amino acids) and a C-terminal extension derivative (424 amino acids), gave rise to two distinct protein bands with molecular masses of approximately 53-55 and 56-58 kDa, respectively, which are approximately 26-30% heavier than those calculated from the deduced amino acid sequences . The differences in molecular mass on SDS-PAGE are thought to be attributed to the acidic nature of the P protein (pI=6.27) and also the different degrees of phosphorylation in the prokaryotic cell . Amino acid sequence comparison of the P protein among the published NDV strains showed that they were highly conserved particularly at the putative phosphorylation sites. Curr Genet, 2002 Aug, 41(5), 291 - 310 Epub 2002 Jul 18. The gene complement for proteolysis in the cyanobacterium Synechocystis sp . PCC 6803 and Arabidopsis thaliana chloroplasts; Sokolenko A et al.; A set of 62 genes that encode the entire peptidase complement of Synechocystis sp . PCC 6803 has been identified in the genome database of that cyanobacterium . Sequence comparisons with the Arabidopsis genome uncovered the presumably homologous chloroplast components inherited from their cyanobacterial ancestor . A systematic gene disruption approach was chosen to individually inactivate, by customary transformation strategies, the majority of the cyanobacterial genes encoding peptidase subunits that are related to chloroplast enzymes . This allowed classification of the peptidases that are required for cell viability or are involved in specific stress responses . The comparative analysis between Synechocystis and Arabidopsis chloroplast peptidases showed that: (1) homologous enzymes that arose by gene duplications in cyanobacteria are functionally diverse and frequently do not complement each other, (2) the chloroplast appears to house a number of distinct peptidase polypeptide chains of cyanobacterial origin (49) which is comparable with a cyanobacterial cell (62) and (3) the peptidase complement in plastids results from a combination of the loss of some cyanobacterial peptidases and the gain or diversification of subclasses of peptidases . This reorganization in the pattern of proteolytic enzymes may reflect distinct environmental and physiological changes between prokaryotic and organellar systems. Genome Biol . 2002 Jun 27;3(7):REVIEWS3010 . Epub 2002 Jun 27. The 14-3-3s; Ferl RJ et al.; Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes . The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client . As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors . There are currently 133 full-length sequences available in GenBank for this highly conserved protein family . A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups . The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences . The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization. Dis Aquat Organ, 2002 Jul 8, 50(2), 137 - 44 Prokaryote infections in the New Zealand scallops Pecten novaezelandiae and Chlamys delicatula; Hine PM et al.; Four intracellular prokaryotes are reported from the scallops Pecten novaezelandiae Reeve, 1853 and Chlamys delicatula Hutton, 1873 . Elongated (1025 x 110 nm), irregular (390 x 200 nm), or toroidal (410 x 200 nm) mollicute-like organisms (M-LOs) occurred free in the cytoplasm in the digestive diverticular epithelial cells of both scallop species . Those in P . novaezelandiae bore osmiophilic blebs that sometimes connected the organisms together, and some had a rod-like protrusion, both of which resemble the blebs and tip structures of pathogenic mycoplasmas . The M-LOs in C . delicatula had a slightly denser core than periphery . Round M-LOs, 335 x 170 nm, occurred free in the cytoplasm of agranular haemocytes in P . novaezelandiae, without apparent harm to the host cell . In P . novaezelandiae, 2 types of highly prevalent (95 to 100%) basophilic inclusions in the branchial epithelium contained Rickettsia-like organisms (R-LOs